Mammalian TNF-α convertases

ABSTRACT

The present invention provides isolated human and bovine TNF-α convertases, nucleic acids and recombinant vectors encoding the same, host cells comprising the nucleic acids and vectors, and methods for making the convertases using the host cells. This invention further provides antibodies and antigen binding fragments thereof which specifically bind to the convertases and are useful for treating medical conditions caused or mediated by TNF-α. Also provided are screening methods for identifying specific inhibitors of mammalian TNF-α convertases, and for identifying nucleic acids encoding such convertases.

This application claims the benefit of U.S. Provisional Application No.60/021,710, filed Jul. 12, 1996.

TECHNICAL FIELD

The present invention relates to mammalian tumor necrosis factor-α(TNF-α) convertase enzymes. More particularly, it relates to bovine,human and other TNF-α convertases, isolated nucleic acids andrecombinant vectors encoding the enzymes, methods for making theenzymes, fragments or fusion proteins thereof using recombinant DNAmethodology or chemical synthesis, and to methods for using the enzymesin screening systems to identify TNF-α convertase inhibitors for thetreatment of various diseases, and nucleic acids encoding a TNF-αconvertase. This invention further relates to antibodies, bothpolyclonal and monoclonal, which specifically bind to the TNF-αconvertases, and to fragments and fusion proteins of the TNF-αconvertases of the invention.

BACKGROUND OF THE INVENTION

TNF-α, also known as cachectin, is a 17 kDa (kilodalton) proteinproduced by cells of the monocyte/macrophage lineage, and by othercells. A variety of biological effects, both beneficial and deleterious,have been attributed to TNF-α. TNF-α is beneficial, e.g., in that it isbelieved to be a part of host anti-tumor defenses. It also producesdetrimental effects, however, including, e.g., cardiovascular (shock,ARDS, capillary leakage syndrome), renal (nephritis, acute tubalnecrosis), and gastrointestinal (ischemia, colitis, hepatic necrosis)effects, and effects on the central nervous system (fever, anorexia,altered pituitary hormone secretion). In view of the foregoing, aconsensus view has developed that TNF-α is a key mediator ofinflammation (including inflammatory diseases such as arthritis) andmammalian responses to injury, invasion by pathogens, and neoplasia.

The biosynthesis of human TNF-α proceeds by way of a membrane-boundprecursor containing 233 amino acid residues Wang et al., Science228:149-154 (1985); Muller et al., Nature 335:265-267 (1987)!, which isprocessed during cellular activation by cleavage of a 76-residue peptideto produce the mature, secreted form of TNF-α. The enzyme(s) responsiblefor this cleavage, called TNF-α convertase, has until the presentinvention been elusive for most mammalian species.

A putative TNF-α convertase, called PR-3, has been isolated and clonedfrom human neutrophils, and it has been suggested that this enzyme canbe used in screens to identify TNF-α convertase inhibitors. SeeInternational Patent Applications Publication Numbers WO 94/00555 and WO95/24501. This enzyme, however, is not believed to be thephysiologically relevant human TNF-α convertase because it is a serineprotease, whereas the relevant enzyme is believed to be ametalloproteinase. Moreover, the source of the serine protease,neutrophils, is not believed to be important in the production of TNF-α,and the serine protease does not cleave the precursor form of TNF-α(proTNF-α) at the point expected for the physiologically relevant humanenzyme.

Mohler et al. Nature 70:218 (1994)! have partially purified anotherTNF-α convertase from the human monocytic cell line THP-1. Thispreparation, however, was very impure, and little could be said aboutthe nature of the TNF-α convertase in the crude protein mixture ofMohler et al.

In view of the important role of TNF-α in many disease processes, thereis a need for agents that can selectively block the biosynthesis ofmature, secreted TNF-α. The search for such agents would be greatlyfacilitated by the availability of substantially pure mammalian TNF-αconvertases.

SUMMARY OF THE INVENTION

The present invention fills the foregoing need by providing materialsand methods for identifying specific inhibitors of TNF-α convertase.More particularly, this invention provides substantially pure mammalianTNF-α convertases capable of converting proTNF-α to the mature, secretedform. This invention further provides isolated or recombinant nucleicacids encoding mammalian TNF-α convertases, and recombinant vectors andhost cells comprising such nucleic acids.

This invention further provides a method for making a mammalian TNF-αconvertase, comprising culturing a host cell comprising a nucleic acidencoding a mammalian TNF-α convertase under conditions in which thenucleic acid is expressed. In some embodiments, the method furthercomprises isolation of the TNF-α convertase from the culture.

This invention also provides polypeptides comprising a fragment of aTNF-α convertase having an amino acid sequence corresponding to thesequence of at least about 8 contiguous residues of the complete enzymesequence. Preferably, the polypeptides comprise at least about 12, morepreferably at least about 20, and most preferably at least about 30 suchresidues.

Still further, this invention provides fusion proteins comprising aTNF-α convertase or a polypeptide thereof covalently linked to a fusionpartner.

The present invention also provides antibodies, both polyclonal in andmonoclonal, that specifically bind to one or more of the TNF-αconvertases or to a polypeptide thereof. Also provided areanti-idiotypic antibodies, both monoclonal and polyclonal, whichspecifically bind to the foregoing antibodies.

This invention still further provides a method of treatment comprisingadministering to a mammal afflicted with a medical condition caused ormediated by TNF-α, an effective amount of an antibody, or anantigen-binding fragment thereof, that specifically binds to a mammalianTNF-α convertase, and pharmaceutical compositions comprising suchantibodies or fragments and pharmaceutically acceptable carriers.

The present invention also provides a method for identifying aninhibitor of a mammalian TNF-α convertase, comprising:

(a) contacting a mammalian TNF-α convertase in the presence of substratewith a sample to be tested for the presence of an inhibitor of theconvertase; and

(b) measuring the rate of cleavage of the substrate;

whereby an inhibitor of the TNF-α convertase in the sample is identifiedby measuring substantially reduced cleavage of the substrate, comparedto what would be measured in the absence of such inhibitor.

In a preferred embodiment, the contacting of the convertase with thesample in the presence of substrate occurs on the surface of a mammalianhost cell comprising one or more nucleic acids encoding a mammalianTNF-α convertase and a substrate of the convertase.

BRIEF DESCRIPTION OF THE FIGURES

The present invention can be more readily understood by reference to thefollowing Description and Examples, and to the accompanying Figures, inwhich:

FIG. 1 is an elution profile from an HPLC column showing DNP-proTNF-αcleavage products;

FIG. 2 is a graphical representation of results from an assay in whichhuman proTNF-α was cleaved by membrane-type matrix metalloproteasesMT-MMP1 and MT-MMP3; and

FIG. 3 is a graphical representation of results from an assay in whichcleavage of human proTNF-α by MT-MMP1 was inhibited by varying amountsof an MMP inhibitor.

DESCRIPTION OF THE INVENTION

All references cited herein are hereby incorporated in their entirety byreference. As used herein, the terms "proteinase" and "protease" areintended to mean the same thing and are used interchangeably. So too arethe terms "assay(s)" and "screen(s)".

Characterization of TNF-α Convertases

The mammalian TNF-α convertases of the present invention arefunctionally characterized by an ability to process, through proteolyticcleavage, the conversion of the membrane-bound form of a precursor formof TNF-α, referred to herein as "proTNF-α", to the soluble, mature form.This processing entails cleavage of the first 76 amino-terminal residuesof the human precursor protein, the entire sequence of which is definedin the Sequence Listing by SEQ ID NO: 1. This sequence, taken from HumanCytokines, B. Aggarwal and J. Gutterman, Eds., 1992, BlackwellScientific Publications, Oxford, pp. 276-277, is in agreement with theSwiss-Prot sequence, Accession Code: Swiss-Prot P01375. There is aconflict, however, with the corresponding GenBank sequence Accession No.M10988; Wang et al., Science 228:149 (1985)!, which has a serine residueat position -14, instead of the phenylalanine residue shown at thatposition in SEQ ID NO: 1.

Regardless of whether one or both of these sequences is correct, asmight be the case with allelic or polymorphic variants, cleavage ofproTNF-α by the TNF-α convertases of this invention occurs at an Ala-Valpeptide bond, resulting in mature human TNF-α having a valine residue atthe amino terminus (i.e., beginning with the Val residue at position 1of SEQ ID NO: 1).

The foregoing cleavage point is different from that observed for theserine protease PR-3 mentioned above. Robache-Gallea et al. J. Biol.Chem. 270:23688 (1995)! have shown that the serine protease cleavesbetween Val¹ and Arg² of SEQ ID NO: 1, thereby producing a mature formof TNF-α having an N-terminal arginine residue.

The mammalian TNF-α convertases of the present invention are furthercharacterized by their presence in cells that make TNF-α. They may bepresent in other cells as well, however, and may even be ubiquitouslyexpressed. Control could be exerted at the level of transcription of theproTNF-α message, or specific controllers of the TNF-α convertases couldbe present in different cell types. It is also not necessary that theTNF-α convertases cleave and process only proTNF-α; they could haveother substrates as well. It also may be that different TNF-αconvertases process proTNF-α in different cell types. Thus, oneconvertase might carry out processing in T and NK cells, while adifferent enzyme might function in macrophages. It therefore may not benecessary that a given TNF-α convertase be present in all cell typesthat make TNF-α.

But apart from the requirement that a TNF-α convertase of this inventionbe present in at least one type of cell that makes TNF-α, whether theother possibilities discussed in the foregoing paragraph are correct ornot is not essential to the invention.

Tryptic digestion followed by amino acid sequencing of a peptide from aTNF-α convertase isolated from bovine spleen revealed that enzyme to befurther characterized by an amino acid sequence comprising a sequencesubstantially as follows:

Met-Asn-Ser-Leu-Leu-Gly/Asp-Ser-Ala-Pro (SEQ ID NO: 2).

This bovine enzyme is further characterized by behavior observed invarious chromatographic systems during purification, as is described indetail in the Example below, and by an apparent molecular weight inSDS-PAGE under reducing conditions of about 65 kDa.

The present invention also encompasses another bovine TNF-α convertaseand enzymes from other mammalian species, including human TNF-αconvertases.

Some general properties of TNF-α convertases as defined in thisinvention are as follows:

(1) Inhibited by ethylenediaminetetraacetic acid (EDTA), dithiothreitol(DTT), 1,10-phenanthroline and/or α-2 macroglobulin.

(2) Not inhibited by serine, cysteine and acid protease inhibitors, suchas 100 μM captopril, 300 μM phosphoramidon, 100 μM thiorphan, 100 μMdichloroisocoumarin (DCI), 1 mM iodoacetic acid (IAA), 1 μg/ml tissueinhibitor of metalloproteases-1 (TIMP-1), 1 μg/ml soybean trypsininhibitor (SBTI), 1 mMmethoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (AAPV) and 100 μMtrans-epoxysuccinyl-L-leucylamino (4-guanidino)-butane (E64; a thiolprotease inhibitor).

(3) Membrane-bound on THP-1 cells and on other monocytic-type cells andcell lines.

(4) Cleave human pro-TNF-α at an Ala-Val peptide bond, to producesoluble, mature TNF-α.

The proteins of the present invention are useful in rational drugdiscovery screens for the identification of compounds that selectivelyblock the conversion of proTNF-α to soluble, mature TNF-α. They havethis utility because when introduced into cells used in the screens,e.g., by transfection of nucleic acids encoding the proteins, theyproduce TNF-α convertase activity which can act on an appropriatesubstrate as described herein. Inhibitors can be identified by measuringinhibition of this activity.

Some Definitions

As used herein, the term "bovine TNF-α convertase" in one embodimentmeans an enzyme having the above-mentioned subsequence and purificationcharacteristics, or a significant fragment of such a protein whichsubstantially retains the proteolytic activity and specificity disclosedherein. In another embodiment, "bovine TNF-α convertase" means bovineADAM 10 GenBank Accession No. Z21961; Wolfsberg et al., J. Cell. Biol.131:275 (1995)!, which the present inventors have surprisingly found isa TNF-α convertase. It also refers to a bovine-derived enzyme exhibitingsimilar enzymatic activity which specifically binds to an antibodyelicited against either of the bovine TNF-α convertases, or to aproteolytically active fragment from one of those enzymes. Suchantibodies typically bind to a bovine or other TNF-α convertase withhigh affinity, e.g., with an affinity constant of at least about 100 nM,usually better than about 30 nM, preferably better than about 10 nM, andmore preferably better than about 3 nM.

Because bovine ADAM 10 is a TNF-α convertase and a comparison of itsamino acid sequence with available sequence information on human ADAM 10shows the two proteins to be 96% homologous, the present inventorsbelieve that human ADAM 10 is also a TNF-α convertase as defined herein.

Surprisingly, the present inventors have also discovered that the humanmembrane-type metalloproteases MT-MMP1 Sato et al., Nature 370:61(1994)!, MT-MMP2 Will et al., Eur. J. Biochem. 231:602 (1995)! andMT-MMP3 Takino et al., J. Biol. Chem. 270:23013 (1995)! are also TNF-αconvertases as defined herein.

The present inventors have further cloned a cDNA encoding a novel humanprotein. When transfected into mammalian cells otherwise incapable ofprocessing human proTNF-α to soluble, mature TNF-α, this proteinproduces such processing. The sequence of this DNA, together with thepredicted amino acid sequence, is substantially as defined in theSequence Listing by SEQ ID NO: 21.

As used herein, the term "polypeptide" means a fragment or segment,e.g., of a TNF-α convertase which comprises a subsequence of thecomplete amino acid sequence of the enzyme containing at least about 8,preferably at least about 12, more preferably at least about 20, andmost preferably at least about 30 or more contiguous amino acidresidues, up to and including the total number of residues in thecomplete enzyme.

The polypeptides of the invention can comprise any part of the completesequence of a TNF-α convertase. Thus, although they could be produced byproteolytic cleavage of an intact enzyme, they can also be made bychemical synthesis or by the application of recombinant DNA technologyand are not limited to polypeptides delineated by proteolytic cleavagesites.

The term "analog(s)" means a TNF-α convertase which has been modified bydeletion, addition, modification or substitution of one or more aminoacid residues in the wild-type enzyme. It encompasses allelic andpolymorphic variants, and also muteins and fusion proteins whichcomprise all or a significant part of a TNF-α convertase, e.g.,covalently linked via a side-chain group or terminal residue to adifferent protein, polypeptide or moiety (fusion partner).

Some amino acid substitutions are preferably "conservative", withresidues replaced with physicochemically similar residues, such asGly/Ala, Asp/Glu, Val/Ile/Leu, Lys/Arg, Asn/Gln and Phe/Trp/Tyr. Analogshaving such conservative substitutions typically retain substantialproteolytic activity. Other analogs, which have non-conservativesubstitutions such as Asn/Glu, Val/Tyr and His/Glu, may substantiallylack proteolytic activity. Nevertheless, such analogs are useful becausethey can be used as antigens to elicit production of antibodies in animmunologically competent host. Because these analogs retain many of theepitopes (antigenic determinants) of the wild-type enzymes from whichthey are derived, many antibodies produced against them can also bind tothe active-conformation or denatured wild-type enzymes. Accordingly, theantibodies can be used, e.g., for the immunopurification or immunoassayof the wild-type enzymes.

Whether a particular analog exhibits convertase activity can bedetermined by routine experimentation as described herein.

Some analogs are truncated variants in which residues have beensuccessively deleted from the amino- and/or carboxyl-termini, whilesubstantially retaining the characteristic proteolytic activity.

Modifications of amino acid residues may include but are not limited toaliphatic esters or amides of the carboxyl terminus or of residuescontaining carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino-terminalamino acid or amino-group containing residues, e.g., lysine or arginine.

This invention also encompasses physical variants having substantialamino acid sequence homology with the amino acid sequences of the TNF-αconvertases or polypeptides. In this invention, amino acid sequencehomology, or sequence identity, is determined by optimizing residuematches and, if necessary, by introducing gaps as required. Homologousamino acid sequences are typically intended to include natural allelic,polymorphic and interspecies variations in each respective sequence.

Typical homologous proteins or peptides will have from 25-100% homology(if gaps can be introduced) to 50-100% homology (if conservativesubstitutions are included), with the amino acid sequence of the TNF-αconvertases. Primate species convertases are of particluar interest.

Observed homologies will typically be at least about 35%, preferably atleast about 50%, more preferably at least about 75%, and most preferablyat least about 85% or more. See Needleham et al., J. Mol. Biol.48:443-453 (1970); Sankoff et al. in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison, 1983,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif., and the University of WisconsinGenetics Computer Group, Madison, Wis.

Glycosylation variants include, e.g., analogs made by modifyingglycosylation patterns during synthesis and processing in variousalternative eukaryotic host expression systems, or during furtherprocessing steps. Particularly preferred methods for producingglycosylation modifications include exposing the TNF-α convertases toglycosylating enzymes derived from cells which normally carry out suchprocessing, such as mammalian glycosylation enzymes. Alternatively,deglycosylation enzymes can be used to remove carbohydrates attachedduring production in eukaryotic expression systems.

Other analogs are TNF-α convertases containing modifications, such asincorporation of unnatural amino acid residues, or phosphorylated aminoacid residues such as phosphotyrosine, phosphoserine or phosphothreonineresidues. Other potential modifications include sulfonation,biotinylation, or the addition of other moieties, particularly thosewhich have molecular shapes similar to phosphate groups.

Analogs of TNF-α convertases can be prepared by chemical synthesis or byusing site-directed mutagenesis Gillman et al., Gene 8:81 (1979);Roberts et al., Nature 328:731 (1987) or Innis (Ed.), 1990, PCRProtocols: A Guide to Methods and Applications, Academic Press, NewYork, N.Y.! or the polymerase chain reaction method PCR; Saiki et al.,Science 239:487 (1988)!, as exemplified by Daugherty et al. NucleicAcids Res. 19:2471 (1991)! to modify nucleic acids encoding the completeenzymes. Adding epitope tags for purification or detection ofrecombinant products is envisioned.

General techniques for nucleic acid manipulation and expression aredescribed generally, e.g., in Sambrook, et al., Molecular Cloning: ALaboratory Manual (2d ed.), 1989, Vols. 1-3, Cold Spring HarborLaboratory. Techniques for the synthesis of polypeptides are described,for example, in Merrifield, J. Amer. Chem. Soc. 85:2149 (1963);Merrifield, Science 232:341 (1986); and Atherton et al., Solid PhasePeptide Synthesis: A Practical Approach, 1989, IRL Press, Oxford.

Still other analogs are prepared by the use of agents known in the artfor their usefulness in cross-linking proteins through reactive sidegroups. Preferred derivatization sites with cross-linking agents arefree amino groups, carbohydrate moieties and cysteine residues.

Substantial retention of proteolytic activity by the foregoing analogsof the TNF-α convertases typically entails retention of at least about50%, preferably at least about 75%, more preferably at least about 80%,and most preferably at least about 90% of the proTNF-α processingactivity and/or specificity of the corresponding wild-type enzyme.

As used herein, the term "isolated nucleic acid" means a nucleic acidsuch as an RNA or DNA molecule, or a mixed polymer, which issubstantially separated from other components that are normally found incells or in recombinant DNA expression systems. These components includebut are not limited to ribosomes, polymerases, serum components, andflanking genomic sequences. The term thus embraces a nucleic acid whichhas been removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules but may, in some embodiments, contain minor heterogeneity.Such heterogeneity is typically found at the ends of nucleic acid codingsequences or in regions not critical to a desired biological function oractivity.

A "recombinant nucleic acid" is defined either by its method ofproduction or structure. Some recombinant nucleic acids are thus made bythe use of recombinant DNA techniques which involve human intervention,either in manipulation or selection. Others are made by fusing twofragments not naturally contiguous to each other. Engineered vectors areencompassed, as well as nucleic acids comprising sequences derived usingany synthetic oligonucleotide process.

For example, a wild-type codon may be replaced with a redundant codonencoding the same amino acid residue or a conservative substitution,while at the same time introducing or removing a nucleic acid sequencerecognition site. Similarly, nucleic acid segments encoding desiredfunctions may be fused to generate a single genetic entity encoding adesired combination of functions not found together in nature. Althoughrestriction enzyme recognition sites are often the target of suchartificial manipulations, other site-specific targets, e.g., promoters,DNA replication sites, regulation sequences, control sequences, or otheruseful features may be incorporated by design. Sequences encodingepitope tags for detection or purification as described above may alsobe incorporated.

A nucleic acid "fragment" is defined herein as a nucleotide sequencecomprising at least about 17, generally at least about 25, preferably atleast about 35, more preferably at least about 45, and most preferablyat least about 55 or more contiguous nucleotides.

This invention further encompasses recombinant DNA molecules andfragments having sequences that are identical or highly homologous tothose described herein. The nucleic acids of the invention may beoperably linked to DNA segments which control transcription,translation, and DNA replication.

"Homologous nucleic acid sequences" are those which when aligned andcompared exhibit significant similarities. Standards for homology innucleic acids are either measures for homology generally used in the artby sequence comparison or based upon hybridization conditions, which aredescribed in greater detail below.

Substantial nucleotide sequence homology is observed when there isidentity in nucleotide residues in two sequences (or in theircomplementary strands) when optimally aligned to account for nucleotideinsertions or deletions, in at least about 50%, preferably in at leastabout 75%, more preferably in at least about 90%, and most preferably inat least about 95% of the aligned nucleotides.

Substantial homology also exists when one sequence will hybridize underselective hybridization conditions to another. Typically, selectivehybridization will occur when there is at least about 55% homology overa stretch of at least about 30 nucleotides, preferably at least about65% over a stretch of at least about 25 nucleotides, more preferably atleast about 75%, and most preferably at least about 90% over about 20nucleotides. See, e.g., Kanehisa, Nucleic Acids Res. 12:203 (1984).

The lengths of such homology comparisons may encompass longer stretchesand in certain embodiments may cover a sequence of at least about 17,preferably at least about 25, more preferably at least about 50, andmost preferably at least about 75 nucleotide residues.

Stringency of conditions employed in hybridizations to establishhomology are dependent upon factors such as salt concentration,temperature, the presence of organic solvents, and other parameters.Stringent temperature conditions usually include temperatures in excessof about 30° C., often in excess of about 37° C., typically in excess ofabout 45° C., preferably in excess of about 55° C., more preferably inexcess of about 65° C., and most preferably in excess of about 70° C.Stringent salt conditions will ordinarily be less than about 1000 mM,usually less than about 500 mM, more usually less than about 400 mM,preferably less than about 300 mM, more preferably less than about 200mM, and most preferably less than about 150 mM. For example, saltconcentrations of 100, 50 and 20 mM are used. The combination of theforegoing parameters, however, is more important than the measure of anysingle parameter. See, e.g., Wetmur et al., J. Mol. Biol. 31:349 (1968).

The term "substantially pure" is defined herein to mean a TNF-αconvertase or other material that is free from other contaminatingproteins, nucleic acids, and other biologicals derived from an originalsource organism or recombinant DNA expression system. Purity may beassayed by standard methods and will typically exceed at least about50%, preferably at least about 75%, more preferably at least about 90%,and most preferably at least about 95% purity. Purity evaluation may bemade on a mass or molar basis.

Antibody Production

Antigenic (i.e., immunogenic) fragments of the TNF-α convertases of thisinvention, which may or may not have enzymatic activity, may similarlybe produced. Regardless of whether they cleave proTNF-α, such fragments,like the complete TNF-α convertases, are useful as antigens forpreparing antibodies, using standard methods, that can bind to thecomplete enzymes. Shorter fragments can be concatenated or attached to acarrier. Because it is well known in the art that epitopes generallycontain at least about five, preferably at least about 8, amino acidresidues Ohno et al., Proc. Natl. Acad. Sci. USA 82:2945 (1985)!,fragments used for the production of antibodies will generally be atleast that size. Preferably, they will contain even more residues, asdescribed above. Whether a given fragment is immunogenic can readily bedetermined by routine experimentation.

Although it is generally not necessary when complete TNF-α convertasesare used as antigens to elicit antibody production in an immunologicallycompetent host, smaller antigenic fragments are preferably firstrendered more immunogenic by cross-linking or concatenation, or bycoupling to an immunogenic carrier molecule (i.e., a macromoleculehaving the property of independently eliciting an immunological responsein a host animal). Cross-linking or conjugation to a carrier moleculemay be required because small polypeptide fragments sometimes act ashaptens (molecules which are capable of specifically binding to anantibody but incapable of eliciting antibody production, i.e., they arenot immunogenic). Conjugation of such fragments to an immunogeniccarrier molecule renders them more immunogenic through what is commonlyknown as the "carrier effect".

Suitable carrier molecules include, e.g., proteins and natural orsynthetic polymeric compounds such as polypeptides, polysaccharides,lipopolysaccharides etc. Protein carrier molecules are especiallypreferred, including but not limited to keyhole limpet hemocyanin andmammalian serum proteins such as human or bovine gammaglobulin, human,bovine or rabbit serum albumin, or methylated or other derivatives ofsuch proteins. Other protein carriers will be apparent to those skilledin the art. Preferably, but not necessarily, the protein carrier will beforeign to the host animal in which antibodies against the fragments areto be elicited.

Covalent coupling to the carrier molecule can be achieved using methodswell known in the art, the exact choice of which will be dictated by thenature of the carrier molecule used. When the immunogenic carriermolecule is a protein, the fragments of the invention can be coupled,e.g., using water soluble carbodiimides such as dicyclohexylcarbodiimideor glutaraldehyde.

Coupling agents such as these can also be used to cross-link thefragments to themselves without the use of a separate carrier molecule.Such cross-linking into aggregates can also increase immunogenicity.Immunogenicity can also be increased by the use of known adjuvants,alone or in combination with coupling or aggregation.

Suitable adjuvants for the vaccination of animals include but are notlimited to Adjuvant 65 (containing peanut oil, mannide monooleate andaluminum monostearate); Freund's complete or incomplete adjuvant;mineral gels such as aluminum hydroxide, aluminum phosphate and alum;surfactants such as hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N',N'-bis(2-hydroxymethyl) propanediamine,methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran,dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides suchas muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.The polypeptides could also be administered following incorporation intoliposomes or other microcarriers.

Information concerning adjuvants and various aspects of immunoassays aredisclosed, e.g., in the series by P. Tijssen, Practice and Theory ofEnzyme Immunoassays, 3rd Edition, 1987, Elsevier, New York. Other usefulreferences covering methods for preparing polyclonal antisera includeMicrobiology, 1969, Hoeber Medical Division, Harper and Row;Landsteiner, Specificity of Serological Reactions, 1962, DoverPublications, New York, and Williams, et al., Methods in Immunology andImmunochemistry, Vol. 1, 1967, Academic Press, New York.

Serum produced from animals immunized using standard methods can be useddirectly, or the IgG fraction can be separated from the serum usingstandard methods such as plasmaphoresis or adsorption chromatographywith IgG-specific adsorbents such as immobilized Protein A.Alternatively, monoclonal antibodies can be prepared.

Hybridomas producing monoclonal antibodies against the TNF-α convertasesof the invention or antigenic fragments thereof are produced bywell-known techniques. Usually, the process involves the fusion of animmortalizing cell line with a B-lymphocyte that produces the desiredantibody. Alternatively, non-fusion techniques for generating immortalantibody-producing cell lines can be used, e.g., virally-inducedtransformation Casali et al., Science 234:476 (1986)!. Immortalizingcell lines are usually transformed mammalian cells, particularly myelomacells of rodent, bovine, and human origin. Most frequently, rat or mousemyeloma cell lines are employed as a matter of convenience andavailability.

Techniques for obtaining antibody-producing lymphocytes from mammalsinjected with antigens are well known. Generally, peripheral bloodlymphocytes (PBLs) are used if cells of human origin are employed, orspleen or lymph node cells are used from non-human mammalian sources. Ahost animal is injected with repeated dosages of the purified antigen(human cells are sensitized in vitro), and the animal is permitted togenerate the desired antibody-producing cells before they are harvestedfor fusion with the immortalizing cell line. Techniques for fusion arealso well known in the art, and in general involve mixing the cells witha fusing agent, such as polyethylene glycol.

Hybridomas are selected by standard procedures, such as HAT(hypoxanthine-aminopterin-thymidine) selection. Those secreting thedesired antibody are selected using standard immunoassays, such asWestern blotting, ELISA (enzyme-linked immunosorbent assay), RIA(radioimmunoassay), or the like. Antibodies are recovered from themedium using standard protein purification techniques Tijssen, Practiceand Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985)!.

Many references are available to provide guidance in applying the abovetechniques Kohler et al., Hybridoma Techniques (Cold Spring HarborLaboratory, New York, 1980); Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985); Campbell, Monoclonal AntibodyTechnology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal HybridomaAntibodies: Techniques and Applications (CRC Press, Boca Raton, Fla.,1982)!. Monoclonal antibodies can also be produced using well knownphage library systems. See, e.g., Huse, et al., Science 246:1275 (1989);Ward, et al., Nature 341:544 (1989).

Antibodies thus produced, whether polyclonal or monoclonal, can be used,e.g., in an immobilized form bound to a solid support by well knownmethods, to purify the TNF-α convertases by immunoaffinitychromatography.

Antibodies against the antigenic fragments can also be used, unlabeledor labeled by standard methods, as the basis for immunoassays of theTNF-α convertases. The particular label used will depend upon the typeof immunoassay. Examples of labels that can be used include but are notlimited to radiolabels such as ³² P, ¹²⁵ I, ³ H and ¹⁴ C; fluorescentlabels such as fluorescein and its derivatives, rhodamine and itsderivatives, dansyl and umbelliferone; chemiluminescers such asluciferia and 2,3-dihydro-phthalazinediones; and enzymes such ashorseradish peroxidase, alkaline phosphatase, lysozyme andglucose-6-phosphate dehydrogenase.

The antibodies can be tagged with such labels by known methods. Forexample, coupling agents such as aldehydes, carbodiimides, dimaleimide,imidates, succinimides, bisdiazotized benzadine and the like may be usedto tag the antibodies with fluorescent, chemiluminescent or enzymelabels. The general methods involved are well known in the art and aredescribed, e.g., in Immunoassay: A Practical Guide, 1987, Chan (Ed.),Academic Press, Inc., Orlando, Fla. Such immunoassays could be carriedout, for example, on fractions obtained during purification of the TNF-αconvertases.

The antibodies of the present invention can also be used to identifyparticular cDNA clones expressing the TNF-α convertases, in expressioncloning systems.

Neutralizing antibodies that bind to the catalytic site of a TNF-αconvertase may also be used as inhibitors to block substrate binding,and hence catalytic activity. This can be done using complete antibodymolecules, or well known antigen binding fragments such as Fab, Fc,F(ab)₂, and Fv fragments.

Definitions of such fragments can be found, e.g., in Klein, Immunology(John Wiley, New York, 1982); Parham, Chapter 14, in Weir, ed.Immunochemistry, 4th Ed. (Blackwell Scientific Publishers, Oxford,1986). The use and generation of antibody fragments has also beendescribed, e.g.: Fab fragments Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985)!, Fv fragments Hochman et al.,Biochemistry 12:1130 (1973); Sharon et al., Biochemistry 15:1591 (1976);Ehrlich et al., U.S. Pat. No. 4,355,023! and antibody half molecules(Auditore-Hargreaves, U.S. Pat. No. 4,470,925). Methods for makingrecombinant Fv fragments based on known antibody heavy and light chainvariable region sequences have further been described, e.g., by Moore etal. (U.S. Pat. No. 4,642,334) and by Pluckthun Bio/Technology 9:545(1991)!. Alternatively, they can be chemically synthesized by standardmethods.

The antibodies and antigen-binding fragments thereof can be usedtherapeutically to block the activity of a TNF-α convertase, and therebyto treat any medical condition caused or mediated by TNF-α. Suchantibodies and fragments are preferably chimeric or humanized, to reduceantigenicity and human anti-mouse antibody (HAMA) reactions. Themethodology involved is disclosed, e.g., in U.S. Pat. No. 4,816,397 toBoss et al. and in U.S. Pat. No. 4,816,567 to Cabilly et al. Furtherrefinements on antibody humanization are described in European Patent451 216 B1.

The dosage regimen involved in a therapeutic application will bedetermined by the attending physician, considering various factors whichmay modify the action of the antibodies or binding fragments, e.g., thecondition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration, and other clinical factors.

Typical protocols for the therapeutic administration of antibodies arewell known in the art and have been disclosed, e.g., by Elliott et al.The Lancet 344:1125 (1994)!, Isaacs et al. The Lancet 340:748 (1992)!,Anasetti et al. Transplantation 54:844 (1992)!, Anasetti et al. Blood84:1320 (1994)!, Hale et al. The Lancet 2:1394 (Dec. 17, 1988)!, QueenScrip 1881:18 (1993)! and Mathieson et al. N. Eng. J. Med. 323:250(1990)!.

Administration of the compositions of this invention is typicallyparenteral, by intraperitoneal, intravenous, subcutaneous, orintramuscular injection, or by infusion or by any other acceptablesystemic method. Administration by intravenous infusion, typically overa time course of about 1 to 5 hours, is preferred.

Often, treatment dosages are titrated upward from a low level tooptimize safety and efficacy. Generally, daily antibody dosages willfall within a range of about 0.01 to 20 mg protein per kilogram of bodyweight. Typically, the dosage range will be from about 0.1 to 5 mgprotein per kilogram of body weight.

Dosages of antigen binding fragments from the antibodies will beadjusted to account for the smaller molecular sizes and possiblydecreased half-lives (clearance times) following administration. Variousmodifications or derivatives of the antibodies or fragments, such asaddition of polyethylene glycol chains (PEGylation), may be made toinfluence their pharmacokinetic and/or pharmacodynamic properties.

It will be appreciated by those skilled in the art, however, that theTNF-α convertase inhibitors of the invention are not limited toneutralizing antibodies or binding fragments thereof. This inventionalso encompasses other types of inhibitors, including small organicmolecules and inhibitory substrate analogs.

One example of a class of small organic molecule inhibitors potentiallyuseful in this invention is a metalloprotease inhibitor designated GI129471, which has been shown to block TNF-α secretion, both in vitro andin vivo McGeehan et al., Nature 370:558 (1994)!. Another such example isN-{D,L-2-(hydroxyaminocarbonyl)methyl!-4-methylpentanoyl}L-3-(2'naphthyl)-alanyl-Lalanine, 2-aminoethyl amide Mohler et al., Nature 370:218 (1994)!, whichhas been shown to protect mice against a lethal dose of endotoxin. Stillanother example of a small organic molecule inhibitor is a compound,designated SCH 43534, which is mentioned in an example below. Thiscompound is a peptide-based hydroxamate inhibitor of collagenasestructurally similar to the foregoing compounds, the inhibitory activityof which validates use of an assay of the invention to identify a TNF-αconvertase inhibitor.

The foregoing small organic molecules are not specific inhibitors of aTNF-α convertase but inhibit other metalloproteases as well. As isdescribed more fully below, specific inhibitors of a TNF-α convertasewhich do not inhibit other metalloproteases can also be identified usingthe methods of this invention if desired.

An "effective amount" of a composition of the invention is an amountthat will ameliorate one or more of the well known parameters thatcharacterize medical conditions caused or mediated by TNF-α. Many suchparameters and conditions have been described, e.g., as in a review byK. J. Tracey in The Cytokine Handbook, Second Edition, A. Thompson, Ed.,1994, Academic Press Ltd., London, UK, pp. 289-304. The references citedby Tracey are also incorporated herein in their entirety by reference.

Although the compositions of this invention could be administered insimple solution, they are more typically used in combination with othermaterials such as carriers, preferably pharmaceutical carriers. Usefulpharmaceutical carriers can be any compatible, non-toxic substancesuitable for delivering the compositions of the invention to a patient.Sterile water, alcohol, fats, waxes, and inert solids may be included ina carrier. Pharmaceutically acceptable adjuvants (buffering agents,dispersing agents) may also be incorporated into the pharmaceuticalcomposition. Generally, compositions useful for parenteraladministration of such drugs are well known; e.g. Remington'sPharmaceutical Science, 17th Ed. (Mack Publishing Company, Easton, Pa.,1990). Alternatively, compositions of the invention may be introducedinto a patient's body by implantable drug delivery systems Urquhart etal., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984)!.

Therapeutic formulations may be administered in many conventional dosageformulation. Formulations typically comprise at least one activeingredient, together with one or more pharmaceutically acceptablecarriers. Formulations may include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. See,e.g., Gilman et al. (eds.) (1990), The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, supra, Easton, Pa.; Avis et al. (eds.) (1993) PharmaceuticalDosage Forms: Parenteral Medications Dekker, N.Y.; Lieberman et al.(eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, N.Y.; andLieberman et al. (eds.) (1990), Pharmaceutical Dosage Forms: DisperseSystems Dekker, N.Y.

The present invention also encompasses anti-idiotypic antibodies, bothpolyclonal and monoclonal, which are produced using the above-describedantibodies as antigens. These antibodies are useful because they maymimic the structures of the proteases.

Protein Purification

The proteins, polypeptides and antigenic fragments of this invention canbe purified by standard methods, including but not limited to salt oralcohol precipitation, preparative disc-gel electrophoresis, isoelectricfocusing, high pressure liquid chromatography (HPLC), reversed-phaseHPLC, gel filtration, cation and anion exchange and partitionchromatography, and countercurrent distribution. Such purificationmethods are well known in the art and are disclosed, e.g., in Guide toProtein Purification, Methods in Enzymology, Vol. 182, M. Deutscher,Ed., 1990, Academic Press, New York, N.Y. More specific methodsapplicable to purification of the bovine TNF-α convertases are describedbelow.

Purification steps can be followed by carrying out assays for TNF-αconvertase activity as described below. Particularly where a convertaseis being isolated from a cellular or tissue source, it is preferable toinclude one or more inhibitors of other proteolytic enzymes is the assaysystem. Such inhibitors include, e.g., 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF), pepstatin, leupeptin, andmethoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (AAPV).

Nucleic Acids and Expression Systems

Nucleic acids encoding the TNF-α convertases or fragments thereof can beprepared by standard methods. For example, DNA can be chemicallysynthesized using, e.g., the phosphoramidite solid support method ofMatteucci et al. J. Am. Chem. Soc. 103:3185 (1981)!, the method of Yooet al. J. Biol. Chem. 764:17078 (1989)!, or other well known methods.This can be done by sequentially linking a series of oligonucleotidecassettes comprising pairs of synthetic oligonucleotides, as describedbelow.

Of course, due to the degeneracy of the genetic code, many differentnucleotide sequences can encode the TNF-α convertases. The codons can beselected for optimal expression in prokaryotic or eukaryotic systems.Such degenerate variants are of course also encompassed by thisinvention.

Moreover, nucleic acids encoding the TNF-α convertases can readily bemodified by nucleotide substitutions, nucleotide deletions, nucleotideinsertions, and inversions of nucleotide stretches. Such modificationsresult in novel DNA sequences which encode antigens having immunogenicor antigenic activity in common with the wild-type enzymes. Thesemodified sequences can be used to produce wild-type or mutant enzymes,or to enhance expression in a recombinant DNA system.

Insertion of the DNAs encoding the TNF-α convertases into a vector iseasily accomplished when the termini of both the DNAs and the vectorcomprise compatible restriction sites. If this cannot be done, it may benecessary to modify the termini of the DNAs and/or vector by digestingback single-stranded DNA overhangs generated by restriction endonucleasecleavage to produce blunt ends, or to achieve the same result by fillingin the single-stranded termini with an appropriate DNA polymerase.

Alternatively, desired sites may be produced, e.g., by ligatingnucleotide sequences (linkers) onto the termini. Such linkers maycomprise specific oligonucleotide sequences that define desiredrestriction sites. Restriction sites can also be generated by the use ofthe polymerase chain reaction (PCR). See, e.g., Saiki et al., Science239:487 (1988). The cleaved vector and the DNA fragments may also bemodified if required by homopolymeric tailing.

Expression of nucleic acids encoding the TNF-α convertases of thisinvention can be carried out by conventional methods in eitherprokaryotic or eukaryotic cells. Although strains of E. coli areemployed most frequently in prokaryotic systems, many other bacteriasuch as various strains of Pseudomonas and Bacillus are know in the artand can be used as well.

Prokaryotic expression control sequences typically used includepromoters, including those derived from the β-lactamase and lactosepromoter systems Chang et al., Nature 198:1056 (1977)!, the tryptophan(trp) promoter system Goeddel et al., Nucleic Acids Res. 8:4057 (1980)!,the lambda P_(L) promoter system Shimatake et al., Nature 292:128(1981)! and the tac promoter De Boer et al., Proc. Natl. Acad. Sci. USA292:128 (1983)!. Numerous expression vectors containing such controlsequences are known in the art and available commercially.

Eukaryotic expression systems typically insect, mammalian or yeast hostcells, for which many expression vectors are known in the art andcommercially available.

Screening Systems and Methods

To identify inhibitors of the TNF-α convertases, the enzymes areemployed in basic screening systems. Essentially, these systems providemethods for bringing together a mammalian TNF-α convertase, anappropriate substrate for the enzyme, and a sample to be tested for thepresence of an inhibitor of the enzyme. If the sample contains such aninhibitor, substantially reduced cleavage of the substrate will beobserved, compared to what would be observed in the absence of aninhibitor, e.g., using a "control" sample containing only buffer.

A basic screening method comprises:

(a) contacting a mammalian TNF-α convertase in the presence of substratewith a sample to be tested for the presence of an inhibitor of theconvertase; and

(b) measuring the rate of cleavage of the substrate;

whereby an inhibitor of the TNF-α convertase in the sample is identifiedby measuring substantially reduced cleavage of the substrate, comparedto what would be measured in the absence of such inhibitor.

"Substantially reduced cleavage" of a substrate by a TNF-α convertaseinhibitor will be observed by measuring less than about 50%, preferablyless than about 25%, more preferably less than about 10%, and mostpreferably less than about 5% of the cleavage measured in the absence ofan inhibitor.

The term "sample" is defined herein to mean any solution, whetheraqueous, organic of some combination of the two, that may contain aTNF-α convertase inhibitor. Examples of samples include but are notlimited to solutions of compounds obtained following organic synthesis,aliquots from purification step fractions, and extracts from cells ortissues, or from other biological or microbial materials.

TNF-α convertase substrates that can be used in the basic assays of theinvention include polypeptides comprising the complete proTNF-αsequence, and truncated variants (polypeptides) thereof, the preferredrequirement being that all substrates contain the specific Ala-Val bond,the cleavage of which characterizes the TNF-α convertases of theinvention. Some examples of such substrates are described below,including a protein (SEQ ID NO: 3) and a polypeptide (SEQ ID NO: 4)substrate. Others are known in the art, such as those disclosed byMohler et al. Nature 370:218 (1995)!. The term "substrate" is definedherein to mean all such materials. Substrates suitable for use in theassays are preferably based on human proTNF-α, although it may bepossible to use substrates from other species.

The substrates can be engineered so that the activity of a TNF-αconvertase causes a positive or negative measurable change in thesubstrate. This may result in a loss or gain of a measurable signal,following cleavage of the substrate.

Any TNF-α convertase can be used in the basic screening methods of thisinvention, although use of a primate or human enzyme is preferred forthe identification of compounds suitable for use as human therapeutics.In connection with the assays, the term "TNF-α convertase" encompassesboth the wild-type variants and analogs, such as truncated orsubstituted variants, as long as they possess substantial proteolyticactivity as defined herein. Use of a wild-type, full-length human enzymeis however preferred. Whether a given analog would be suitable for usein an assay of the invention can readily be determined through routineexperimentation, using the disclosed methods.

Those skilled in the art will appreciate that there are many ways amammalian TNF-α convertase could be brought together with a substrateand a test sample to identify an inhibitor, and all such methods arewithin the scope of this invention. Nevertheless, in a preferredembodiment, a mammalian cell system is employed in which one or morenucleic acids encoding a mammalian TNF-α convertase and a substrate aretransfected into a host cell. These nucleic acids can be contained in asingle recombinant vector or in two, as is the case in an Example below.

Particularly preferred mammalian host cells for use in the foregoingsystem inherently lack or have minimal ability to cleave proTNF-α to themature, secreted form. Examples of such a cell are the 293 humanembryonic kidney cell line and clones derived therefrom. The 293 line isavailable from the American Type Culture Collection, Rockville, Md.,under Accession No. ATCC CRL 1573. A clone derived from the 293 line,designated 293EBNA, is available from Invitrogen.

TNF-α convertase inhibitors identified in the basic screens of thisinvention may be suitable for therapeutic administration, although theymay also inhibit other metalloproteases. If it is desired to identify aspecific inhibitor of a TNF-α convertase, i.e., one that will notinhibit the activity of other, more general metalloproteases, that canbe done using another embodiment of the present invention.

As used herein, the term "'specific inhibitor of a TNF-α convertase" isdefined to mean an inhibitor which blocks the proteolytic activity of aTNF-α convertase but does not inhibit the activity of collagenase orother matrix-degrading metalloproteases.

The following is a summary of some matrix-degrading metalloproteases,including some names by which they have been called and some of theirsubstrates:

    ______________________________________                                        Enzyme Names        Substrates                                                ______________________________________                                        Interstitial Collagenase (MMP-1)                                                                  Collagens I, II, III, VII and X                           72-kDa Gelatinase (MMP-2)                                                                         Collagens IV, V, VII and X                                Stromelysin (MMP-3) Collagens III, IV, V and IX                               Uterine Metalloproteinase                                                                         Gelatins I, III, IV and V                                 (MMP-7)                                                                       Neutrophil Collagenase (MMP-8)                                                                    Collagens I, II and III                                   92-kDa Gelatinase (MMP-9)                                                                         Collagens IV and V                                        Stromelysin-2 (MMP-10)                                                                            Gelatins I, III, IV and V                                 ______________________________________                                    

More information on the names and substrates, and on the properties ofthe above-mentioned matrix-degrading metalloproteases, can be found in areview by Woessner FASEB J. 5:2145 (1991)!.

To identify a specific inhibitor of a TNF-α convertase, the screeningmethods of the invention further comprise:

(a) contacting a matrix-degrading metalloprotease in the presence ofsubstrate with an inhibitor of a TNF-α convertase; and

(b) measuring the rate of cleavage of the substrate;

whereby a specific inhibitor of a TNF-α convertase is identified bymeasuring substantially undiminished cleavage of the substrate, comparedto what would be measured in the absence of such inhibitor.

In a preferred embodiment, a mammalian cell system is employed in whichone or more nucleic acids encoding a matrix-degrading metalloproteaseand a substrate are transfected into a host cell. These nucleic acidscan be contained in a single recombinant vector or in two.

"Substantially undiminished cleavage" of a substrate by a specificinhibitor of a TNF-α convertase will be observed by measuring at leastabout 75%, preferably at least about 90%, more preferably at least about95%, and most preferably at least about 99% of the cleavage measured inthe absence of such an inhibitor.

Molecular Cloning and Expression

The present invention provides methods for cloning bovine TNF-αconvertase and corresponding enzymes from other mammalian species.Briefly, Southern and Northern blot analysis can be carried out toidentify cells from other species expressing genes encoding the TNF-αconvertases. Complementary DNA (cDNA) libraries can be prepared bystandard methods from mRNA isolated from such cells, and degenerateprobes or PCR primers based on the amino acid sequence informationprovided herein can be used to identify clones encoding a TNF-αconvertase.

Alternatively, expression cloning methodology can be used to identifyparticular clones encoding a TNF-α convertase. An antibody preparationwhich exhibits cross-reactivity with TNF-α convertases from a number ofmammalian species may be useful in monitoring expression cloning.

Preferably, a co-transfection system described more fully below is usedto identify clones capable of cleaving proTNF-α to the mature, secretedform. Selected clones can then be amplified, and cDNA isolated from themcan be inserted into vectors suitable for expression in prokaryotic oreukaryotic expression systems.

Briefly, this method for identifying a nucleic acid encoding a mammalianTNF-α convertase comprises:

(a) culturing a mammalian host cell comprising a first recombinantexpression vector comprising a nucleic acid encoding a TNF-α convertasesubstrate and a second recombinant expression vector comprising anucleic acid that is to be tested to determine whether it encodes amammalian TNF-α convertase, under conditions in which expression occurs;and

(b) measuring the rate of cleavage of the substrate;

whereby a nucleic acid encoding a mammalian TNF-α convertase isidentified by measuring substantially increased cleavage of thesubstrate, compared to what would be measured in the absence of suchnucleic acid.

Preferably the TNF-α convertase substrate used is proTNF-α, although anyof the other substrates mentioned herein could be used instead.

In the context of this invention, "substantially increased cleavage" ofthe substrate will be observed by measuring at least about 5 times more,preferably at least about 10 times more, more preferably at least about25 times more, and most preferably at least about 50 times more cleavageof the substrate than would occur in the absence of a nucleic acidencoding a mammalian TNF-α convertase.

However identified, clones encoding TNF-α convertases from variousmammalian species can be isolated and sequenced, and the coding regionscan be excised and inserted into an appropriate vector.

Recombinant expression vectors in this invention are typicallyself-replicating DNA or RNA constructs comprising nucleic acids encodingone of the TNF-α convertases, usually operably linked to suitablegenetic control elements that are capable of regulating expression ofthe nucleic acids in compatible host cells. Genetic control elements mayinclude a prokaryotic promoter system or a eukaryotic promoterexpression control system, and typically include a transcriptionalpromoter, an optional operator to control the onset of transcription,transcription enhancers to elevate the level of mRNA expression, asequence that encodes a suitable ribosome binding site, and sequencesthat terminate transcription and translation. Expression vectors alsomay contain an origin of replication that allows the vector to replicateindependently of the host cell.

Vectors that could be used in this invention include microbial plasmids,viruses, bacteriophage, integratable DNA fragments, and other vehicleswhich may facilitate integration of the nucleic acids into the genome ofthe host. Plasmids are the most commonly used form of vector but allother forms of vectors which serve an equivalent function and which are,or become, known in the art are suitable for use herein. See, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985 andSupplements, Elsevier, N.Y., and Rodriguez et al. (eds.), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, 1988, Buttersworth,Boston, Mass.

Suitable host cells for expressing nucleic acids encoding the TNF-αconvertases include prokaryotes and higher eukaryotes. Prokaryotesinclude both gram negative and positive organisms, e.g., E. coli and B.subtilis. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express the TNF-α.convertases include but are not limited to those containing the lacpromoter (pUC-series); trp promoter (pBR322-trp); Ipp promoter (thepIN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters suchas ptac (pDR540). See Brosius et al., "Expression Vectors EmployingLambda-, trp-, lac-, and Ipp-derived Promoters", in Rodriguez andDenhardt (eds.) Vectors: A Survey of Molecular Cloning Vectors and TheirUses, 1988, Buttersworth, Boston, pp. 205-236.

Higher eukaryotic tissue culture cells are preferred hosts for therecombinant production of enzymatically active TNF-α convertases.Although any higher eukaryotic tissue culture cell line might be used,including insect baculovirus expression systems, mammalian cells arepreferred. Transformation or transfection and propagation of such cellshas become a routine procedure. Examples of useful cell lines includeHeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney(BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS)cell lines. Expression vectors for such cell lines usually include anorigin of replication, a promoter, a translation initiation site, RNAsplice sites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Representative examples of suitable expression vectorsinclude pCDNA1, pCD Okayama et al., Mol. Cell Biol. 5:1136 (1985)!,pMC1neo Poly-A Thomas et al., Cell 51:503 (1987)!, pUC19, pREP8,pSVSPORT and derivatives thereof, and baculovirus vectors such as pAC373 or pAC 610.

EXAMPLES

The present invention can be illustrated by the following examples.

Unless otherwise indicated, percentages given below for solids in solidmixtures, liquids in liquids, and solids in liquids are on a wt/wt,vol/vol and wt/vol basis, respectively. Sterile conditions weregenerally maintained during cell culture.

General Methods

Standard methods were used, as described, e.g., in Maniatis et al.,Molecular Cloning: A Laboratory Manual, 1982, Cold Spring HarborLaboratory, Cold Spring Harbor Press; Sambrook et al., MolecularCloning: A Laboratory Manual, (2d ed.), Vols 1-3, 1989, Cold SpringHarbor Press, N.Y.; Ausubel et al., Biology, Greene PublishingAssociates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements),Current Protocols in Molecular Biology, Greene/Wiley, New York; Innis etal. (eds.) PCR Protocols: A Guide to Methods and Applications, 1990,Academic Press, N.Y.

In vitro assays for TNF-α Convertase Activity

For the assay of convertase activity associated with or isolated frommembrane preparations, a protein-based assay was carried out essentiallyas described by Mohler et al., Nature 370:218 (1995).

Briefly, a peptide-tagged recombinant human TNF-α protein substrate(Flag-TNF-α) was cloned and expressed in E. coli. The protein waspurified by affinity chromatography using an M2 (anti-Flag)-Sepharosecolumn (Kodak), and by ion exchange chromatography using a BIOCADequipped with an HQ10 column.

The amino acid sequence of the protein substrate is defined in theSequence Listing by SEQ ID NO: 3, concerning which the following shouldbe noted. Amino acid residues 2-9 comprise the "Flag" sequence. Residues10 and 11 are a Gly-Ser connector following the Flag which were added toaccommodate a restriction site used in construction. The histidine atposition 12 corresponds to the histidine at position -25 of SEQ ID NO:1, after which the two sequences are identical to the carboxyl termini.Thus, fifty residues of the normal leader sequence of human proTNF-αhave been deleted from this substrate. Since the deleted region is notessential for use as a substrate in an assay of this invention, othertruncations containing deletion of more or fewer residues could be usedas well.

To test for convertase activity, a membrane protein sample (12 μg) wasmixed (in 12 μl) with 2 ng of ¹²⁵ I-polypeptide substrate (approximately50,000 cpm) in the presence of inhibitors of other proteolytic enzymese.g., 200 μM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride(AEBSF), 2 μM pepstatin, 200 μM leupeptin, 1 mMmethoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (AAPV)!, and with orwithout 2.5 mM EDTA. Following incubation at 37° C. overnight, sampleswere fractionated in a 16% sodium dodecylsulfate (SDS) gel and subjectedto SDS polyacrylamide gel electrophoresis SDS-PAGE; Laemmli, Nature227:680 (1970)!. The gels were dried, and radioactivity was detected byautoradiography.

Examination of the gels revealed intensified bands from the samplesincubated without EDTA, at the position expected for the 17 kDa humanTNF-α cleavage product.

A polypeptide-based assay for TNF-α convertase activity was also carriedout essentially using the method of Mohler et al., supra. Briefly, anamino-terminal dinitrophenylated peptide substrate, amidated at thecarboxyl terminus (SEQ ID NO: 4) was synthesized and purified usingstandard procedures. Dinitrophenylated polypeptides corresponding tocleavage products expected for TNF-α convertase (a dinitrophenylatedpeptide having the sequence of residues 1-6 of SEQ ID NO: 4) and forother membrane proteases were also synthesized.

To test for convertase activity, a membrane protein sample was mixed in50 μl with 1 μg peptide substrate in the presence of inhibitors (0.2 mMAEBSF, 2 μM pepstatin, 200 μM leupeptin, 1 mM AAPV). Followingincubation at 37° C. for from 60 minutes to overnight, the protein wasprecipitated by cold 5% trichloroacetic acid (TCA), 20% acetonitrile,and the soluble peptide fraction was applied to a YMC 120 angstrom C-18ODS-AQ column (4.6×100 mm; YMC, Inc.). The column was elutedisocratically at a flow rate of 1 ml/min using 40% acetonitrile plus0.06% trifloroacetic acid. The elution of DNP-peptide was monitored at360 nm using a Waters 625 LC system.

The results of a typical assay are shown in FIG. 1, wherein thepositions of the uncut peptide and an expected convertase cleavageproduct (DNP-APLAQA) are shown. Of course, other cleavage productsattributable to the activities of other proteases present in the crudemembrane sample were also observed; some of their elution positions arealso shown in FIG. 1.

Preparation and Sequencing of Bovine TNF-α Convertases

Bovine spleen purchased from Pel-Freeze was cut into small pieces,washed in cold PBS, and then shredded using a Black & Decker POWER PROfood processor. The tissue was resuspended in lysis buffer 20 mM Tricine(N- 2-Hydroxy-1,1-bis(hydroxymethyl)-ethyl! glycine), pH 7.8, 8%sucrose! containing 0.1% PMSF, then homogenized using a BrinkmannPOLYTRON tissue homogenizer. Cell debris was removed by twocentrifugations at 8,000×g, and the membranes were isolated byultracentrifugation at 60,000×g. The isolated membranes were washed in10 mM Hepes, pH 7.5, then resuspended and frozen at -20° C. until used.

The membrane fraction was thawed and resuspended to a concentration of 8mg/ml protein in Buffer A (20 mM Tris, pH 7.5, 1 mM MgSO₄, 10 mM NaCl,10 μM ZnSO₄) plus 2% Brij 35 (23 lauryl ether), and incubated at 4° C.for 30 minutes. The membranes were collected by ultracentrifugation at60,000×g, then resuspended to 4 mg/ml in buffer A plus 2% Lubrol(polyethylene glycol monododecyl ether). The insoluble protein wasremoved by ultracentrifugation at 60,000×g.

The Brij-insoluble, lubrol-solubilized membrane protein fraction wasadjusted to 0.3M NaCl and applied to a chelating Sepharose columncharged with nickel sulfate. The column was washed and eluted with washbuffer (Buffer A with 0.3M NaCl and 0.1% octyl glucoside) plus 50 mMimidazole. The eluate was concentrated by ultrafiltration, then appliedto a S300 sieving column equilibrated in buffer A plus 0.1%octylglucoside. A retained fraction (corresponding to a molecular weightof approximately 60,000) was pooled, adjusted to 0.3M NaCl and appliedto a wheat germ agglutinin column.

The retained fraction was eluted with 0.5M N-acetyl glucosamine inBuffer A with 0.5M NaCl, dialyzed against 1 mM sodium phosphate buffer,pH 7, and applied to a hydroxyapatite column. The unbound fraction waspassed over an HQ-10 ion exchange column (Perseptive Biosystems), andeluted with a linear gradient of from 0 to 500 mM NaCl. Fractionscontaining TNF-α convertase activity were pooled for furthercharacterization.

The final protein fraction (approximately 10 μg) of the first bovineTNF-α convertase was applied to an 8% polyacrylamide gel in SDS-glycinebuffer (Novex). Following electrophoresis, the gel was stained for 8minutes in 10% acetic acid/50% methanol containing 0.1% Coomassie blue,then destained for 3.5 hours in three changes of 10% acetic acid/50%methanol. The polypeptide was excised and subjected to in situ trypticcleavage, peptide isolation and microsequencing using standard methods.

This enzyme was a bovine TNF-α convertase, amino acid sequences of whichare disclosed above.

The purification of bovine ADAM 10 was carried out by subjecting bovinespleen to the procedures described above to the point of application tothe wheat germ agglutinin column. Thereafter, the retained fraction waseluted with 0.5M N-acetyl glucosamine, dialyzed against 1 mM sodiumphosphate buffer, pH 7, and applied to an hydroxyapatite column. A boundfraction from that column was passed over an HQ-10 ion exchange column.

The unbound fraction, containing the TNF-α convertase activity(approximately 10 μg) was subjected to electrophoresis an 8%polyacrylamide gel using SDS-glycine buffer (Novex) with dithiothreitol.Following electrophoresis, the fractionated proteins wereelectophoretically transferred to an IMMOBILON filter membrane in 10 mMCAPS (3- Cyclohexylamino!-1-propanesulfonic acid) buffer, pH 10, with10% methanol. The protein band was visualized by staining the membrane0.1% Ponceau S in 40% methanol, 10% acetic acid, excised, and subjectedto in situ tryptic cleavage, peptide isolation and microsequencing,including N-terminal analysis, using standard methods.

Cloning of Human MT-MMP1, MT-MMP2, MT-MMP3, MMP7, MMP12 and Bovine ADAM10

Human MT-MMP1 cDNA was cloned from THP-1 cell (ATCC TIB 202) total RNA,which was converted to single-stranded DNA using a GibcoBRL SUPERSCRIPTPreamplification System (Catalog #18089-011) and an oligo dT primer.This DNA was then used directly for PCR using primers designated #5261(SEQ ID NO: 5; 5' forward primer) and #5271 (SEQ ID NO: 6; 3' reverseprimer). PCR conditions were: 94° C., 30 seconds/60° C., 30 seconds/72°C., 2 minutes, for 30 cycles. The PCR product was cut with KpnI/HindIIIand ligated into similarly cut pSVSPORT (GibcoBRL).

Cloning of human MT-MMP2 was initiated by isolating total RNA from THP-1cells and preparing single-stranded DNA as described above. The DNA wasthen subjected to a two-step PCR protocol to obtain the full-lengthMT-MMP2 cDNA as follows.

First, two PCR reactions were run that encompassed overlapping front andback halves of MT-MMP2. One reaction was set up using primers designated#B5295GD (SEQ ID NO: 7; 5' forward) and "reverse internal" (SEQ ID NO:8; internal 3'). A second PCR reaction was set up using primers #B5296GD(SEQ ID NO: 9; 3' reverse) and "forward internal" (SEQ ID NO: 10;internal 5'). PCR conditions were as described above. The product ofeach of these reactions was isolated by agarose gel electrophoresis.

In the second step, the two products from the PCR reactions were mixedwith PCR primers B5295GD and B5296GD, and PCR was performed under thesame conditions as described above. The product from this reaction wascut with EcoRI/XbaI, isolated by agarose gel electrophoresis, and clonedinto vector pSRαSPORT that had been cut with the same restrictionenzymes.

Human MT-MMP3 was cloned as described for MT-MMP1 but from aorta polyA⁺RNA (Clontech) using PCR primers designated #5322 (SEQ ID NO: 11; 5'forward primer) and #5323 (SEQ ID NO: 12; 3' reverse primer). The PCRproduct was cut with KpnI/HindIII, isolated, and cloned into vectorpSRαSPORT that had been cut with the same restriction enzymes.

Similarly, human MMP7 (matrilysin) was cloned from human testis poly A⁺RNA (Clontech) and using PCR primers #5367 (SEQ ID NO: 13; 5' forwardprimer) and #5369 (SEQ ID NO: 14; 3' reverse primer). The PCR productwas cut with KpnI/HindIII, isolated, and cloned into vector pSRαSPORTthat had been cut with the same restriction enzymes.

Human MMP12 (macrophage metalloelastase) was similarly cloned from humanaorta polyA⁺ RNA using PCR primers #A0698H03 (SEQ ID NO: 15; 5' forwardprimer) and #A0698H08 (SEQ ID NO: 16; 3' reverse primer). The PCRproduct was cut with KpnI/XbaI and, following isolation, cloned intosimilarly-cut vector pSRαSPORT.

Bovine ADAM 10 was cloned as described above from 5 μg of total RNAisolated from bovine spleen poly A⁺ RNA (Clontech). The resultingsingle-stranded DNA was then used for PCR, using primers havingsequences defined in the Sequence listing by SEQ ID NO: 17 (5' forwardprimer) and SEQ ID NO: 18 (3' reverse primer). PCR conditions were asdescribed above, and the PCR product was digested with KpnI and HindIIIand ligated into similarly-digested vector pCEP4 (Invitrogen).

Construction of a Human proTNF-α Expression Vector

Human proTNF-α cDNA can be cloned from total RNA isolated from LPS(lipopolysaccharide)-stimulated THP- 1 cells and converted tosingle-stranded DNA as described above. This DNA can then be used forPCR, e.g., using primers designed to introduce BamHI cleavage sites intothe PCR product. The sequences of suitable 5' and 3' primers are definedin the Sequence Listing by SEQ ID NO: 19 and SEQ ID NO: 20,respectively. The PCR product is then cut with BamHI to produce aninsert that can be cloned into a BamHI-cleaved expression vector.

A similar insert was ligated into pUC19 (New England Biolabs) that hadbeen cut with BamHI, to produce a vector designated pUCTNF. VectorpUCTNF was then cut with SalI/HindIII, and a fragment retaining thecoding region for proTNF-α was ligated into a vector designatedpSRαSPORT that had cut with the same restriction enzymes. VectorpSRαSPORT had previously been constructed as follows.

pSVSPORT 1 (GibcoBRL) was cut with ClaI/PstI to remove the SV promoterand treated with Klenow polymerase to fill in the ClaI overhang andproduce a blunt end. A fragment containing the SRα promoter and SV40 tantigen was obtained following cleavage of plasmid pDSRG (ATCC 68233;International Patent Application Publication No. WO 91/01078) withHindIII/PstI and filling of the HindIII overhang with Klenow polymerase.This fragment was then ligated into the cut pSVSPORT 1 to producepSRαSPORT.

Cloning of a Novel Human Protein

Vector pSRαSPORT was cleaved using NotI/SalI, and a 1.5 kb stuffer cDNAfragment was ligated into the cut vector. The stuffer cDNA fragment,which was a neomycin resistance gene, was prepared by digesting plasmidPMC1neo Poly A (Stratagene, Catalog No. 213201) with XhoI/SalI, and thesmall fragment was isolated and ligated into SalI-digested pSL1190(Pharmacia, catalog No. 27-4386). The stuffer fragment was released bydigesting the resulting vector pSC1190-Neo with SalI, and the 1.5 kbfragment was isolated.

The construct incorporating the stuffer fragment was cleaved usingNotI/SalI, and the linearized vector was separated from the insert cDNAby agarose gel electrophoresis. The cleaved vector was then repurifiedin a second agarose electrophoresis gel. The pure cleaved vector DNA wasisolated from the agarose gel using GELZYME (Invitrogen), following therecommended conditions. This vector was used to clone library cDNA.

Messenger RNA was prepared by treating Mono Mac-6 cellsZiegler-Heitbrock et al., Int. J. Cancer 41:456 (1988)! withlipopolysaccharide at 1 μg/ml for 18 hours, and with PMA at 10 ng/ml forone hour prior to RNA isolation. Total RNA was prepared from these cellsusing the guanidine thiocyanate method (Sambrook et al., supra, pp.7.19-7.22). The cells were collected by centrifugation, resuspended inguanidine thiocyanate (Gibco BRL) with 2.5 grams of N-laurylsarcosinesodium salt (Sigma), 5 drops of anti-foam A (Sigma) and 0.75 ml of2-mercaptoethanol (Biorad).

The lysate was layered over an equal volume of 5.7M cesium chloridesolution and centrifuged in an SW41 rotor for 24 hours at 25,000 rpm.The resulting total RNA pellet was washed with ethanol, resuspended insterile water, and treated with DNAse by incubating 500 μg of total RNAper ml in buffer containing 5 units of RQ1 DNAse I (Promega), 400 unitsof RNAsin (Promega), 10 mM MgCl₂, and 5 mM DTT at 37° C. for 30 min. Thesolution was treated with an equal volume of 1:1 phenol/chloroformsolution, and the RNA was precipitated with ethanol. PolyA⁺ mRNA wasisolated from the total RNA using the OLIGOTEX mRNA isolation system(Qiagen Inc.).

Five micrograms of mRNA were used to synthesize cDNA following theprotocols in the SUPERSCRIPT Plasmid System for cDNA synthesis andplasmid cloning (Gibco BRL), with the following modifications. Followingsecond strand synthesis, the cDNA was phenol/chloroform extracted,ethanol precipitated, and then treated with T4 DNA Polymerase (PharmaciaLKB), following the manufacturer's instructions. Following NotIdigestion, the resuspended cDNA was subjected to electrophoresis in 1%SEAPLAQUE GTG agarose (FMC) and visualized by ethidium bromide staining.The portion of the gel from 2 kb to 13 kb was excised and digested withGELZYME (Invitrogen).

The resulting size-enriched cDNA was ligated with the NotI/SalI-cleavedpSRαSport vector overnight using a 2:1 vector/insert concentrationratio. The ligation mixture was then extracted with phenol/chloroform,precipitated with ethanol, and electroporated into ELECTROMAX DH10Bcells (Gibco BRL) under the prescribed conditions. The cells were platedout at a density of about 1000 colonies per plate, with a total ofaround 7×10⁵ colonies for the entire library.

Cells from each plate were collected in 1.5 ml Luria broth. An aliquot(500 μl) of each pool was mixed with 250 μl of 80% (v/v) glycerol, andstored at -20° C. The remaining cells were collected by centrifugation,and plasmid DNA isolated using the QIAWELL 8 Ultra Plasmid Kit (Qiagen)following the recommended procedures. Final DNA preparation was elutedfrom the Qiawell resin with two 100 μl aliquots of 10 mM Tris pH8.

Positives from an initial 800 plasmid pools were identified using thecell transfection assay described below and then split into pools of 50colonies per plate, and DNA was prepared as described above. Positivesfrom secondary assays were then spread onto Petri plates, and 144individual colonies were picked. DNA was prepared from each clone asdescribed above, and all positive pools were reconfirmed in duplicate byretransfection and ELISA at each step before proceeding.

Plasmid DNA was sequenced by the Taq DyeDeoxy™ Terminator CycleSequencing kit and method (Perkin-Elmer/Applied Biosystems) in twodirections using an Applied Biosystems Model 373A DNA Sequencer.Sequence alignment was performed using ABI SEQED Analysis and SequenceNavigator software. The results are shown in SEQ ID NO: 21, whichprovides the complete open reading frame nucleotide sequence togetherwith the predicted amino acid sequence.

When co-transfected into 293EBNA cells with the vector encoding humanproTNF-α as described below, the novel cDNA caused the production ofsoluble, mature TNF-α. This activity was inhibited by SCH 43534.

Cell Transfection TNF-α Convertase Assay System

To develop an effective cell-based assay system, it was first necessaryto identify host cells that substantially lacked TNF-α convertaseactivity. This was accomplished by transiently transfecting a number ofcell types with an expression vector encoding a substrate such as humanproTNF-α, and then assaying for the expected cleavage products, as isexplained more fully below.

It thus was established that although COS cells possess significantTNF-α convertase activity, the human embryonic kidney cell line 293 anda clone derived therefrom, designated 293EBNA, lacked the ability tocleave transfected human proTNF-α. That was true even though all of thecells when transfected with a vector expressing a human growth hormoneas a control secreted that product. For that reason, 293/293EBNA cellsare preferred transfection hosts for the assay described below.

This assay system was established using nucleic acids encoding knownmembrane-type matrix metalloproteases, some of which possess TNF-αconvertase activity as defined herein. Using this system in conjunctionwith a vector(s) encoding one of the specific human membrane-type matrixmetalloproteases or bovine ADAM 10, and human proTNF-α, specific TNF-αconvertase inhibitors can be identified.

Moreover, the same system can also be used to identify other nucleicacids encoding other mammalian TNF-α convertases as well. That can beaccomplished by substituting nucleic acids from cDNA or other librariesfor the nucleic acids encoding the exemplary matrix metalloproteases,and observing TNF-α convertase activity expressed thereby.

To demonstrate use of this basic assay system, host cells weretransfected in one of two ways. In one method, human 293EBNA cells(Invitrogen) at 5×10⁶ /0.25 ml of RPMI with 10% fetal bovine serum (FBS)were placed in a 0.4 cm electroporation-cuvette with 5 μg total DNA. Thecells were electroporated using a GENE PULSER (BioRad) at 200 V, 960μFd, and 100 ohms. After recovering for 5 minutes, the cells werediluted into 15 ml of medium and placed in a tissue culture flask at 37°C., 5% CO₂.

In a second method, transfections were carried out using Lipofectin(GibcoBRL). DNA (2 μg total) was mixed with 10 μl of Lipofectin in 200μl of serum free medium (Opti-MEM, GibcoBRL) at room temperature for 15minutes. Then 600 μl of Opti-MEM was added and the mixture added to 10⁶293EBNA cells. After 4 hours at 37° C., 200 μl of 50% FBS were added,and the supernatant was collected 24-48 hours later for analysis. Forsmaller numbers of cells the conditions were scaled down proportionally.

The Lipofectin method was also used to assay for bovine ADAM 10activity, whereby 1 μl of Lipofectin was diluted into 10 μl of OPTI-MEMmedium and allowed to stand at room temperature for 30 minutes. Thesolution was mixed with 10 μl of OPTI-MEM containing 50 ng ofproTNFα-SRαSPORT with or without 100 ng of ADAM 10-pCEP4, and allowed tostand at room temperature for 15 minutes. Sixty μl of OPTI-MEM wereadded, and the entire 80 μl were used to replace the spent growth mediumin the seeded wells. After incubation at 37° C., 5% CO₂ for 5 hours, 20μl of DME containing 50% fetal bovine serum were added to the well.Following incubation at 37° C., 5% CO₂ for 20 hours, the medium wasassayed for TNFα production.

To detect convertase activity, enzyme-linked immunosorbant assay (ELISA)was carried out by diluting a human TNF-α capture antibody (Pharmingen#18631D) to 1 μg/ml in 0.1M NaHCO₃, pH 8.2, and coating the solutiononto a 96 well Nunc MAXISORP microtiter plate at 100 μl/well overnightat 4° C. The wells were then blocked with 200 μl/well of PBS containing10% FBS and 0.1% azide and stored at 4° C. until used for assay.

Immediately prior to use, the microtiter wells were washed with PBScontaining 0.05% Tween 20 (polyoxyethylenesorbitan monolaurate). Samplesand standards were diluted in PBS containing 10% FBS, added at 100μl/well, and incubated at 37° C. for 2 hours. After washing as describedabove, 100 μl of biotinylated anti-TNF-α (Pharmingen #18642D) were addedat 1 μg/ml in PBS with 10% FBS and incubated for 45 minutes at 22° C.After washing twice with PBS and Tween 20, streptavidin-HRP (BioSource)was added at a 1:50 dilution, 100 μl/well in PBS with 10% FBS, andincubated for 30 minutes at 22° C. After washing three times with PBSand Tween 20, ABTS substrate (KP Labs) was added at 100 μl/well, andcolor development was stopped at an appropriate time determined byvisual inspection by addition of 100 μl/well of 1% SDS. The plates wereread at 405 nm using a Molecular Devices microtiter plate reader.

Cleavage of proTNF-α by Human MT-MMP1, MT-MMP2 and MT-MMP3 and by BovineADAM 10

DNA encoding MT-MMP1 or MT-MMP3 described above was cloned intoexpression vector pREP8 (Invitrogen) and co-transfected with the vectorencoding human proTNF-α into 293EBNA cells. Following an assay carriedout as described above, it was observed that both of the matrixmetalloproteases caused cleavage of the proTNF-α. This is evident inFIG. 2, where the results produced using proTNF-α alone (Bar 1),proTNF-α plus MT-MMP1 (Bar 2), and proTNF-α plus MT-MMP3 (Bar 3) areshown. Similar activity was not shown by MMP7 or by MMP12.

It was further found that as little as 6 pg/transfection of MT-MMP1produced a clear secreted TNF-α signal by ELISA. This was determined byvarying the amount of MT-MMP1 vector in the presence of a constantamount of proTNF-α (50 ng/transfection) and empty pREP8 vector (100ng/transfection). Thus, this system is useful for identifying otherTNF-α convertases by expression cloning.

Although the data are not shown, similar results were obtained forMT-MMP2

Similar results were also obtained using bovine ADAM 10.

TNF-α Convertase Inhibitor Assay

To demonstrate use of the transfection assay system to detect inhibitorsof a human TNF-α convertase, 293EBNA cells were co-transfected withexpression vectors encoding human proTNF-α and MT-MMP1 as describedabove, and assayed in the presence or absence of varying amounts of anMMP inhibitor, designated SCH 43534, which had previously been shown toblock the release of TNF-α from activated human THP-1 cells.

The results are shown in FIG. 3 for proTNF-α alone (Bar 1), proTNF-αplus MT-MMP1 (Bar 2), proTNF-α plus MT-MMP1 plus 1 μM SCH 43534 (Bar 3),and proTNF-α plus MT-MMP1 plus 10 μM SCH 43534 (Bar 4).

Similar results were obtained using bovine ADAM 10. It thus is clearthat the present assay can be used to detect inhibitors of TNF-αconvertase activity as defined herein.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, together with the full scope ofequivalents to which such claims are entitled.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 699 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The residue at position -14 of the                     sequence is a phenylanine in the Swiss-Prot sequence,                         Accession Code Swiss-Prot P01375, and as a serine in                          the GenBank sequence, Accession Number M10988.                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      ATGAGCACTGAAAGCATGATCCGGGACGTGGAGCTGGCCGAGGAGGCG48                            MetSerThrGluSerMetIleArgAspValGluLeuAlaGluGluAla                              75-70-65                                                                      CTCCCCAAGAAGACAGGGGGGCCCCAGGGCTCCAGGCGGTGCTTGTTC96                            LeuProLysLysThrGlyGlyProGlnGlySerArgArgCysLeuPhe                              60-55-50-45                                                                   CTCAGCCTCTTCTCCTTCCTGATCGTGGCAGGCGCCACCACGCTCTTC144                           LeuSerLeuPheSerPheLeuIleValAlaGlyAlaThrThrLeuPhe                              40-35-30                                                                      TGCCTGCTGCACTTTGGAGTGATCGGCCCCCAGAGGGAAGAGTTCCCC192                           CysLeuLeuHisPheGlyValIleGlyProGlnArgGluGluXaaPro                              25-20- 15                                                                     AGGGACCTCTCTCTAATCAGCCCTCTGGCCCAGGCAGTCAGATCATCT240                           ArgAspLeuSerLeuIleSerProLeuAlaGlnAlaValArgSerSer                              10-51                                                                         TCTCGAACCCCGAGTGACAAGCCTGTAGCCCATGTTGTAGCAAACCCT288                           SerArgThrProSerAspLysProValAlaHisValValAlaAsnPro                              5101520                                                                       CAAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTC336                           GlnAlaGluGlyGlnLeuGlnTrpLeuAsnArgArgAlaAsnAlaLeu                              253035                                                                        CTGGCCAATGGCGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCA384                           LeuAlaAsnGlyValGluLeuArgAspAsnGlnLeuValValProSer                              404550                                                                        GAGGGCCTGTACCTCATCTACTCCCAGGTCCTCTTCAAGGGCCAAGGC432                           GluGlyLeuTyrLeuIleTyrSerGlnValLeuPheLysGlyGlnGly                              556065                                                                        TGCCCCTCCACCCATGTGCTCCTCACCCACACCATCAGCCGCATCGCC480                           CysProSerThrHisValLeuLeuThrHisThrIleSerArgIleAla                              707580                                                                        GTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAAGAGCCCC528                           ValSerTyrGlnThrLysValAsnLeuLeuSerAlaIleLysSerPro                              859095100                                                                     TGCCAGAGGGAGACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAG576                           CysGlnArgGluThrProGluGlyAlaGluAlaLysProTrpTyrGlu                              105110115                                                                     CCCATCTATCTGGGAGGGGTCTTCCAGCTGGAGAAGGGTGACCGACTC624                           ProIleTyrLeuGlyGlyValPheGlnLeuGluLysGlyAspArgLeu                              120125130                                                                     AGCGCTGAGATCAATCGGCCCGACTATCTCGACTTTGCCGAGTCTGGG672                           SerAlaGluIleAsnArgProAspTyrLeuAspPheAlaGluSerGly                              135140145                                                                     CAGGTCTACTTTGGGATCATTGCCCTG699                                                GlnValTyrPheGlyIleIleAlaLeu                                                   150155                                                                        (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: There is an ambiquity in the residue                   at position 6 ofthe sequence, which may be either a                           Gly or an Asp.                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      MetAsnSerLeuLeuXaaSerAlaPro                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 193 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      MetAspTyrLysAspAspAspAspLysGlySerHisPheGlyValIle                              151015                                                                        GlyProGlnArgGluGluSerProArgAspLeuSerLeuIleSerPro                              202530                                                                        LeuAlaGlnAlaValArgSerSerSerArgThrProSerAspLysPro                              354045                                                                        ValAlaHisValValAlaAsnProGlnAlaGluGlyGlnLeuGlnTrp                              505560                                                                        LeuAsnArgArgAlaAsnAlaLeuLeuAlaAsnGlyValGluLeuArg                              65707580                                                                      AspAsnGlnLeuValValProSerGluGlyLeuTyrLeuIleTyrSer                              859095                                                                        GlnValLeuPheLysGlyGlnGlyCysProSerThrHisValLeuLeu                              100105110                                                                     ThrHisThrIleSerArgIleAlaValSerTyrGlnThrLysValAsn                              115120125                                                                     LeuLeuSerAlaIleLysSerProCysGlnArgGluThrProGluGly                              130135140                                                                     AlaGluAlaLysProTrpTyrGluProIleTyrLeuGlyGlyValPhe                              145150155160                                                                  GlnLeuGluLysGlyAspArgLeuSerAlaGluIleAsnArgProAsp                              165170175                                                                     TyrLeuAspPheAlaGluSerGlyGlnValTyrPheGlyIleIleAla                              180185190                                                                     Leu                                                                           (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The residue at position 1 is Ser                       that has been dinitrophenylated at the α-amino group;                   that at position 12 is Arg, the carboxyl terminus of                          which has been amidated.                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      XaaProLeuAlaGlnAlaValArgSerSerSerXaa                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      TAGCGGTACCGCCCACACTGCCCGGCTGACC31                                             (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      CAACAAGCTTACCACCACCTTGCTGACACTGGTC34                                          (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      ACTAGAATTCAGAGCATGGGCAGCGACCCGAG32                                            (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      GCCCTTGAACACGAACATCTCC22                                                      (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      ACTATCTAGAGCCCCCTGAGCACCGTTAGCA31                                             (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     GCCACAGCCTACCCAGCCTCTC22                                                      (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     TAGCGGTACCACAGTTCACTATGATCTTACTCACA35                                         (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     TAGCAAGCTTAGCCTGCTCCTAGCTAGGAAACAGC35                                         (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     ACTAGGTACCATGCGACTCACCGTGCTG28                                                (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     CACAAGCTTGCTCACCGCCCCGCCGCCCT29                                               (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     CTACGGTACCACAATGAAGTTTCTTCTAATAC32                                            (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     CTTCTCTAGACTAACAACCAAACCAGCT28                                                (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     CTTCCGGGTACCCGGAAGATGGTGTTGCTGAGAGTG36                                        (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     CGTTAAAAGCTTTTAACGTCTCATGTGTCCCATCTG36                                        (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     ACCGGGATCCATGAGCACTGAAAGCATGATC31                                             (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     GTCTGGATCCGAATCCCAGGTTTCGAAGTGGT32                                            (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2352 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     ATGCCACATACTTTGTGGATGGTGTGGGTCTTGGGGGTCATCATCAGC48                            MetProHisThrLeuTrpMetValTrpValLeuGlyValIleIleSer                              151015                                                                        CTCTCCAAGGAAGAATCCTCCAATCAGGCTTCTCTGTCTTGTGACCGC96                            LeuSerLysGluGluSerSerAsnGlnAlaSerLeuSerCysAspArg                              202530                                                                        AATGGTATCTGCAAGGGCAGCTCAGGATCTTTAAACTCCATTCCCTCA144                           AsnGlyIleCysLysGlySerSerGlySerLeuAsnSerIleProSer                              354045                                                                        GGGCTCACAGAAGCTGTAAAAAGCCTTGACCTGTCCAACAACAGGATC192                           GlyLeuThrGluAlaValLysSerLeuAspLeuSerAsnAsnArgIle                              50556065                                                                      ACCTACATTAGCAACAGTGACCTACAGAGGTGTGTGAACCTCCAGGCT240                           ThrTyrIleSerAsnSerAspLeuGlnArgCysValAsnLeuGlnAla                              707580                                                                        CTGGTGCTGACATCCAATGGAATTAACACAATAGAGGAAGATTCTTTT288                           LeuValLeuThrSerAsnGlyIleAsnThrIleGluGluAspSerPhe                              859095                                                                        TCTTCCCTGGGCAGTCTTGAACATTTAGACTTATCCTATAATTACTTA336                           SerSerLeuGlySerLeuGluHisLeuAspLeuSerTyrAsnTyrLeu                              100105110                                                                     TCTAATTTATCGTCTTCCTGGTTCAAGCCCCTTTCTTCTTTAACATTC384                           SerAsnLeuSerSerSerTrpPheLysProLeuSerSerLeuThrPhe                              115120125130                                                                  TTAAACTTACTGGGAAATCCTTACAAAACCCTAGGGGAAACATCTCTT432                           LeuAsnLeuLeuGlyAsnProTyrLysThrLeuGlyGluThrSerLeu                              135140145                                                                     TTTTCTCATCTCACAAAATTGCAAATCCTGAGAGTGGGAAATATGGAC480                           PheSerHisLeuThrLysLeuGlnIleLeuArgValGlyAsnMetAsp                              150155160                                                                     ACCTTCACTAAGATTCAAAGAAAAGATTTTGCTGGACTTACCTTCCTT528                           ThrPheThrLysIleGlnArgLysAspPheAlaGlyLeuThrPheLeu                              165170175                                                                     GAGGAACTTGAGATTGATGCTTCAGATCTACAGAGCTATGAGCCAAAA576                           GluGluLeuGluIleAspAlaSerAspLeuGlnSerTyrGluProLys                              180185190                                                                     AGTTTGAAGTCAATTCAGAATGTAAGTCATCTGATCCTTCATATGAAG624                           SerLeuLysSerIleGlnAsnValSerHisLeuIleLeuHisMetLys                              195200205210                                                                  CAGCATATTTTACTGCTGGAGATTTTTGTAGATGTTACAAGTTCCGTG672                           GlnHisIleLeuLeuLeuGluIlePheValAspValThrSerSerVal                              215220225                                                                     GAATGTTTGGAACTGCGAGATACTGATTTGGACACTTTCCATTTTTCA720                           GluCysLeuGluLeuArgAspThrAspLeuAspThrPheHisPheSer                              230235240                                                                     GAACTATCCACTGGTGAAACAAATTCATTGATTAAAAAGTTTACATTT768                           GluLeuSerThrGlyGluThrAsnSerLeuIleLysLysPheThrPhe                              245250255                                                                     AGAAATGTGAAAATCACCGATGAAAGTTTGTTTCAGGTTATGAAACTT816                           ArgAsnValLysIleThrAspGluSerLeuPheGlnValMetLysLeu                              260265270                                                                     TTGAATCAGATTTCTGGATTGTTAGAATTAGAGTTTGATGACTGTACC864                           LeuAsnGlnIleSerGlyLeuLeuGluLeuGluPheAspAspCysThr                              275280285290                                                                  CTTAATGGAGTTGGTAATTTTAGAGCATCTGATAATGACAGAGTTATA912                           LeuAsnGlyValGlyAsnPheArgAlaSerAspAsnAspArgValIle                              295300305                                                                     GATCCAGGTAAAGTGGAAACGTTAACAATCCGGAGGCTGCATATTCCA960                           AspProGlyLysValGluThrLeuThrIleArgArgLeuHisIlePro                              310315320                                                                     AGGTTTTACTTATTTTATGATCTGAGCACTTTATATTCACTTACAGAA1008                          ArgPheTyrLeuPheTyrAspLeuSerThrLeuTyrSerLeuThrGlu                              325330335                                                                     AGAGTTAAAAGAATCACAGTAGAAAACAGTAAAGTTTTTCTGGTTCCT1056                          ArgValLysArgIleThrValGluAsnSerLysValPheLeuValPro                              340345350                                                                     TGTTTACTTTCACAACATTTAAAATCATTAGAATACTTGGATCTCAGT1104                          CysLeuLeuSerGlnHisLeuLysSerLeuGluTyrLeuAspLeuSer                              355360365370                                                                  GAAAATTTGATGGTTGAAGAATACTTGAAAAATTCAGCCTGTGAGGAT1152                          GluAsnLeuMetValGluGluTyrLeuLysAsnSerAlaCysGluAsp                              375380385                                                                     GCCTGGCCCTCTCTACAAACTTTAATTTTAAGGCAAAATCATTTGGCA1200                          AlaTrpProSerLeuGlnThrLeuIleLeuArgGlnAsnHisLeuAla                              390395400                                                                     TCATTGGAAAAAACCGGAGAGACTTTGCTCACTCTGAAAAACTTGACT1248                          SerLeuGluLysThrGlyGluThrLeuLeuThrLeuLysAsnLeuThr                              405410415                                                                     AACATTGATATCAGTAAGAATAGTTTTCATTCTATGCCTGAAACTTGT1296                          AsnIleAspIleSerLysAsnSerPheHisSerMetProGluThrCys                              420425430                                                                     CAGTGGCCAGAAAAGATGAAATATTTGAACTTATCCAGCACACGAATA1344                          GlnTrpProGluLysMetLysTyrLeuAsnLeuSerSerThrArgIle                              435440445450                                                                  CACAGTGTAACAGGCTGCATTCCCAAGACACTGGAAATTTTAGATGTT1392                          HisSerValThrGlyCysIleProLysThrLeuGluIleLeuAspVal                              455460465                                                                     AGCAACAACAATCTCAATTTATTTTCTTTGAATTTGCCGCAACTCAAA1440                          SerAsnAsnAsnLeuAsnLeuPheSerLeuAsnLeuProGlnLeuLys                              470475480                                                                     GAACTTTATATTTCCAGAAATAAGTTGATGACTCTACCAGATGCCTCC1488                          GluLeuTyrIleSerArgAsnLysLeuMetThrLeuProAspAlaSer                              485490495                                                                     CTCTTACCCATGTTACTAGTATTGAAAATCAGTAGGAATGCAATAACT1536                          LeuLeuProMetLeuLeuValLeuLysIleSerArgAsnAlaIleThr                              500505510                                                                     ACGTTTTCTAAGGAGCAACTTGACTCATTTCACACACTGAAGACTTTG1584                          ThrPheSerLysGluGlnLeuAspSerPheHisThrLeuLysThrLeu                              515520525530                                                                  GAAGCTGGTGGCAATAACTTCATTTGCTCCTGTGAATTCCTCTCCTTC1632                          GluAlaGlyGlyAsnAsnPheIleCysSerCysGluPheLeuSerPhe                              535540545                                                                     ACTCAGGAGCAGCAAGCACTGGCCAAAGTCTTGATTGATTGGCCAGCA1680                          ThrGlnGluGlnGlnAlaLeuAlaLysValLeuIleAspTrpProAla                              550555560                                                                     AATTACCTGTGTGACTCTCCATCCCATGTGCGTGGCCAGCAGGTTCAG1728                          AsnTyrLeuCysAspSerProSerHisValArgGlyGlnGlnValGln                              565570575                                                                     GATGTCCGCCTCTCGGTGTCGGAATGTCACAGGATAGCACTGGTGTCT1776                          AspValArgLeuSerValSerGluCysHisArgIleAlaLeuValSer                              580585590                                                                     GGCATGTGCTGTGCTCTGTTCCTGCTGATCCTGCTCACGGGGGTCCTG1824                          GlyMetCysCysAlaLeuPheLeuLeuIleLeuLeuThrGlyValLeu                              595600605610                                                                  TGCCACCGTTTCCATGGCCTGTGGTATATGAAAATGATGTGGGCCTGG1872                          CysHisArgPheHisGlyLeuTrpTyrMetLysMetMetTrpAlaTrp                              615620625                                                                     CTCCAGGCCAAAAGGAAGCCCAGGAAAGCTCCCAGCAGGAACATATGT1920                          LeuGlnAlaLysArgLysProArgLysAlaProSerArgAsnIleCys                              630635640                                                                     TATGATGCATTTGTTTCTTACAGTGAGCGGGATGCCTACTGGGTGGAG1968                          TyrAspAlaPheValSerTyrSerGluArgAspAlaTyrTrpValGlu                              645650655                                                                     AACCTAATGGTCCAGGAGCTGGAGAACTTCAATCCCCCCTTCAAGTTG2016                          AsnLeuMetValGlnGluLeuGluAsnPheAsnProProPheLysLeu                              660665670                                                                     TGTCTTCATAAGCGGGACTTCATTCCTGGCAAGTGGATCATTGACAAT2064                          CysLeuHisLysArgAspPheIleProGlyLysTrpIleIleAspAsn                              675680685690                                                                  ATCATTGACTCCATTGAAAAGAGCCACAAAACTGTCTTTGTGCTTTCT2112                          IleIleAspSerIleGluLysSerHisLysThrValPheValLeuSer                              695700705                                                                     GAAAACTTTGTGAAGAGTGAGTGGTGCAAGTATGAACTGGACTTCTCC2160                          GluAsnPheValLysSerGluTrpCysLysTyrGluLeuAspPheSer                              710715720                                                                     CATTTCCGTCTTTTTGATGAGAACAATGATGCTGCCATTCTCATTCTT2208                          HisPheArgLeuPheAspGluAsnAsnAspAlaAlaIleLeuIleLeu                              725730735                                                                     CTGGAGCCCATTGAGAAAAAAGCCATTCCCCAGCGCTTCTGCAAGCTG2256                          LeuGluProIleGluLysLysAlaIleProGlnArgPheCysLysLeu                              740745750                                                                     CGGAAGATAATGAACACCAAGACCTACCTGGAGTGGCCCATGGACGAG2304                          ArgLysIleMetAsnThrLysThrTyrLeuGluTrpProMetAspGlu                              755760765770                                                                  GCTCAGCGGGAAGGATTTTGGGTAAATCTGAGAGCTGCGATAAAGTCC2352                          AlaGlnArgGluGlyPheTrpValAsnLeuArgAlaAlaIleLysSer                              775780785                                                                     __________________________________________________________________________

What is claimed is:
 1. An isolated bovine TNF-α convertase characterizedby:(a) an amino acid sequence comprising a sequence defined by SEQ IDNO: 2, (b) an apparent molecular weight in SDS-PAGE of about 65 kDa, and(c) an ability to cleave human proTNF-α to produce soluble, mature humanTNF-α.
 2. An isolated protein comprising an amino acid sequence encodedby the nuceotide sequence defined by SEQ ID NO:
 21. 3. A method foridentifying an inhibitor of the TNF-α convertase of claim 1,comprising:(a) contacting the TNF-α convertase in the presence ofsubstrate with a sample to be tested for the presence of an inhibitor ofthe TNF-α convertase; and (b) measuring the rate of cleavage of thesubstrate;whereby an inhibitor of the TNF-α convertase in the sample isidentified by measuring substantially reduced cleavage of the substrate,compared to what would be measured in the absence of such inhibitor. 4.The method of claim 3 which further comprises:(a) contacting amatrix-degrading metalloprotease in the presence of substrate with aninhibitor of a TNF-α convertase; and (b) measuring the rate of cleavageof the substrate;whereby a specific inhibitor of a TNF-α convertase isidentified by measuring substantially undiminished cleavage of thesubstrate, compared to what would be measured in the absence of suchinhibitor.
 5. The method of claim 4 in which the matrix-degradingmetalloprotease is selected from the group consisting of MMP-1, MMP-2,MMP-3, MMP-7, MMP-8, MMP-9 and MMP-10.
 6. The method of claim 3 in whichsaid contacting occurs on the surface of a mammalian host cellcomprising one or more nucleic acids encoding TNF-α convertase of claim1, and a substrate of the convertase.
 7. The method of claim 4 in whichsaid contacting occurs on the surface of a mammalian host cellcomprising one or more nucleic acids encoding a matrix-degradingmetalloprotease and a substrate of the protease.
 8. The method of claim6 in which the host cell is from human embryonic kidney cell line 293 ora clone thereof.
 9. The method of claim 6 in which the substrate ishuman proTNP-α.